Campylobacter glycans and glycopeptides

ABSTRACT

Multiple strains and species of  Campylobacter  were found to share a common glycan moitie which is present in a plurality of surface-exposed glycoproteins. This glycan has the formula: GalNAc-a1,4-GalNAc-a1,4-[Glc-β1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-Bac, wherein Bac is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose. This glycan and immunologically active fragments of it have use as vaccines against  campylobacter  infection in humans and animals. As well, antibodies which specifically bind these compounds may be provided. Such antibodies and vaccines may be used to prevent or neutralize  campylobacter  infections in livestock thereby preventing this pathogen from entering the human food chain. The glycan may be linked to one or more amino acids to form a glycopeptide. As well, a method for determining the glycan structure of an intact glycoprotein consists of subjecting a sample to high resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) spectroscopy.

FIELD OF THE INVENTION

The invention is in the field of biologically active glycoproteins andglycan moieties of glyocoproteins as well as vaccines, antibodies andantibody fragments useful for combating campylobacter bacteria.

BACKGROUND OF THE INVENTION

Campylobacter is a significant food borne pathogen in livestockproducts, including poultry. It is thus desirable to provide strategiesfor combating its deleterious effects in humans. Elimination of thesepathogens from livestock can serve as a means to reduce the incidence ofinfection in humans and reduce disease in farm animals that areaffected. Glycosylated proteins and in particular certain glycanmoieties of these compounds represent viable targets for immunologicstrategies to reduce or eliminate the presence of these organisms inlivestock, in order to reduce the risk of human contamination fromeating or handling animal products as well as contamination by fecalshedding of bacteria from livestock manure. Glycoproteins and theirfragments such as glycan moieties may also provide a basis for vaccinesand antibody preparation which target campylobacter infections.

It is also desirable to provide research tools having generalapplication for studying protein glycosylation. Glycosylation ofproteins was once considered to be specifically a eukaryotic phenomenon,but it is now clear that it is widespread in both the Archaea andEubacteria domains (1,2). Glycosidic linkages of both the N- and O-types have been identified in a diverse group of prokaryotic organismswith a preponderance of N-linked sugars apparent in the Archaea whilelinkage units of the O-type predominate in glycoproteins identified thusfar in the Eubacteria (1,2). In addition, bacterial N- and O- linkagesare formed with a wider range of sugars than those observed ineukaryotic glycoproteins.

The present inventors have discovered that certain glycopeptides andgylcan moieties form an effective basis for vaccines and antibodiesagainst multiple strains of campylobacter bacteria, by virtue of theirubiquitous presence on the surface of these bacteria in an invariant (ornearly invariant) form across multiple strains and species. This glycanis also present as a component of a plurality of surface glycoproteinsof campylobacter thus enhancing its value as an immunologic target.

Recently a gene locus was identified in the enteric pathogenCampylobacter jejuni, which appears to be involved in the glycosylationof multiple proteins. This provided the first evidence of a pathway forwide-spread protein glycosylation in a gram-negative bacterium (3).Mutagenesis of genes within this locus, termed pgl (for proteinglycosylation), resulted in loss of immunogenicity in multiple proteins.The glycan moieties of these proteins were also shown to be recognizedby antisera from experimentally infected human volunteers (3). Removalof the glycan moieties by pgl mutation resulted in decreased adherenceand invasion in vitro and loss of mouse colonization in vivo (4),suggesting that protein glycosylation influences the virulenceproperties of this organism.

The present inventors have identified and characterizedpost-translational modifications of proteins in C. jejuni strain NCTC11168, the strain for which the whole genome sequence has been describedby Parkhill et al. (6). Among the proteins giving rise to multiple spotson 2D gels was PEB3, or Cj0289c, a major antigenic protein of C. jejunifirst described by Pei et al. (7). When purified and analysed by 1DSDS-PAGE, it revealed two bands with a mass difference of ˜1500 Da, bothof which had N-terminal sequences corresponding to authentic PEB3.Concurrent with our observations on PEB3, Linton et al. (8) identifiedtwo putative glycoproteins from C. jejuni by use of the GalNAc-specificlectin, soybean agglutinin, one of which was PEB3, and the other aputative periplasmic protein Cj1670c, which they named CgpA. The authorsalso observed a number of other putative glycoproteins, based upon theirability to bind to the lectin, but these were not identified.Furthermore, protein binding to the lectin was also affected bymutagenesis of genes in the pgl locus. In addition, we have shown thatmutation of a gene pglB, whose homology to the STT3 subunit of theN-linked oligosaccharyltransferase of Saccharomyces cerevisae suggesteda role in glycoprotein biosynthesis (3,9), specifically affects theglycosylation of the identified glycoproteins.

Moreover, carbohydrates are implicated in a variety of functions in alldomains of life. While in general there is high degree of variance ofglycoproteins and their glycan moieties in antigenic surface glycansbetween bacterial strains and related species, this is not always thecase and such exceptions present suitable excellent candidates forantibody or vaccine-based strategies for eliminating bacteria. Inparticular, one such carbohydrate is the glycan moiety of campylobacterglycoproteins. Our recent efforts to characterize the glycome of theimportant foodborne pathogen Campylobacter jejuni, has led to theelucidation of all surface glycan structures, namely,lipooligosaccharide (LOS), capsular polysaccharide (CPS), N-linkedglycans, and O-linked glycans (5, 12-15)

It has been shown by the present inventors that the heptasaccharide,which is described in this specification, is common to at least severalCampylobacter species and numerous strains including species that areimportant as human and veterinary pathogens, and is a component ofmultiple glycoproteins including Cj nos. 0114, 0200c, 0289c, 0367c andothers. This glycan moiety is also strongly immunogenic and as such thisglycan (and related fragements and glycopeptides) is a good candidatefor use as a vaccine for active immunization against multiple strainsand species of campylobacter and as the basis for antibodies or antibodyfragments suitable for targeting of campylobacter in a human or inlivestock.

As used herein “livestock” includes mammals and poultry.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a glycan which may existeither in isolation or linked to an oligopeptide, an amino acid and/oran immunogenic conjugate.

In accordance with one aspect, the invention provides a compoundcomprising a heptasaccharide of formula I:GalNAc-a1,4-GalNAc-a1,4-[Glc-β1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-Bac,wherein Bac is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose. Formula Ialso includes immunologically active fragments of the compound describedabove. By “immunologically active” is meant that such fragment maycomprise the active component of a vaccine against campylobacter, orthat antibodies or antibody fragments may be provided which specificallybind to such a compound and to neutralize campylobacter in an organismin which the same has been administered. These glycans may be providedin isolated form or linked to an oligopeptide or amino acid to form anovel glycopeptide. The amino acid may comprise asparagine to form anN-linked glycopeptide. These glycans or glycopeptides may be derivedfrom or isolated and purified from a gram negative bacterium. Thisglycoprotein glycan moiety can also provide the basis for therapeuticpreparations and methods as discussed below.

In accordance with yet another aspect, the invention provides a methodof detecting a glycan moiety of a bacterial glycoprotein, the methodcomprising subjecting said sample to high resolution magic anglespinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy. Thismethod allows for the detection of the glycan moiety directly form itssource without the need of purifying the protein.

In accordance with a further aspect, the invention provides apharmaceutical composition which comprises a heptasaccharide of formulaI and a physiologically acceptable carrier. Preferably, thepharmaceutical compositions may further comprise an immunogenicconjugate bound to the glycan or glycopeptide or fragment thereof and/oran immunostimulant. Such pharmaceutical compositions are useful asvaccines for immunizing a human or an animal against diseases caused bycampylobacter pathogens. Such diseases are for example gastro-intestinalinfections, Guillain Barré GBS and Miller Fisher syndromes, arthritisand bacteremia. Such vaccines may be administered via one or moreinjections or by another medically acceptable method.

In accordance with another aspect, the invention provides an antibody oran antigen-binding antibody fragment that interacts and specificallybinds with the glycan moiety of Formula I. Such antibodies or fragmentsmay neutralize one or more Campylobacter species when administered to ananimal or human recipient. Such antibodies can be obtained by variousknown methods including being isolated from the serum of an animal thathas been previously immunized with a heptasaccharide or a glycopeptideas described above or preparing murine monoclonal antibodies directedagainst such compounds. Another means is via recombinant DNA techniques,for example by obtaining such an antibody or antibody fragment bycloning a library set of domain antibody (“dAb”) genes previouslyisolated from a camelid, and then expressed in a bacteriophage library,panning for and then expressing in a bacteriophage a sequence dAbshaving an affinity for the heptasaccharide, glycopeptide or fragmentthereof. A preferred camelid is selected from Camelus bactriamus,Camelus dromaderius, Lama pPaccos, Lama ggGlama and Lama vAlternatively,another method could involve screening bacteriophage libraries ofsingle-chain antibody fragments (scFv)Vicugna. The invention alsoprovides a pharmaceutical composition comprising the antibody or itsantigen-binding fragment as defined above, and a physiologicallyacceptable carrier. Such pharmaceutical composition can be used as atherapeutic agent in an animal or a human.

Suitable antibody fragments include Fab fragments (although it will benoted that since Camelid species produce a unique type of antibody whichdoes not contain light chains, conventional terminology such as “Fab”may be modified) including fragments which are prepared in isolation bythe above techniques or by other conventional techmiques and notdirectly derived from a whole antibody.

In accordance with yet another aspect, the invention provides a methodof reducing the presence of campylobacter bacteria in livestock. Themethod comprises administering to livestock the antibody orantigen-binding antibody fragment that interacts and binds with theglycan moiety of Formula I. Such antibody when administered insufficient quantity neutralizes all or substantially all pathogeniccampylobacter organisms from the animal's system or preventscolonization by the bacteria thereby preventing the animal frominfecting humans, either by eating or handling an animal product or viacontamination from animal waste. The administration may consist offeeding the livestock, preferably poultry, with feed or water containingthe antibody or antigen-binding fragment. Eliminating the pathogens orreducing their presence in livestock constitutes a way of preventing thepathogens from reaching humans who are therefore prevented from diseasescaused by these pathogens. Thus, the invention also provides for amethod of preventing campylobacter infections caused by campylobacterpathogens in a human, the method comprising removing the pathogens fromlivestock by delivering to the livestock the antibody or antigen-bindingfragment thereof as defined above.

The antibodies or antibody fragments may be either added to animal feedor water or alternatively may be present as a result of a geneticmodification of an animal feed plant which permits such a plant toexpress the antibody or antibody fragment.

In accordance with still another aspect, the invention provides a methodof treating a disease caused by campylobacter pathogens in a human or ananimal, the method comprising administering the antibody orantigen-binding fragment thereof as defined above to the human. Theantibody or its antigen-binding fragment can be used in the preparationof a medicament for treating an animal or a human for a disease causedby campylobacter pathogens.

In accordance with another aspect, the invention provides for method ofpreventing ground water contamination of campylobacter pathogens whicharises as a result of fecal shedding of the bacteria by livestockfollowed by entry of the fecal matter into a water supply. The methodcomprises eliminating or reducing the presence of the pathogens fromlivestock by administering to the livestock the antibody orantigen-binding fragment thereof as defined above. This also constitutesa way of preventing the pathogens from reaching humans, thereforepreventing them from diseases caused by these pathogens.

The antibodies and antibody fragments of the present invention may alsobe used as a diagnostic tool to detect the presence of campylobacter inhumans and animals, as well as in samples drawn from the environmentsuch as water or manure samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Purification and analysis of PEB3. a) Cation-exchangechromatography of a glycine extract on MonoS column. PEB3 containingfractions were identified by N-terminal sequencing of SDS-PAGE bands,pooled as indicated, dialyzed and freeze-dried. b) Re-fractionation ofpooled material on MonoS column using a shallower gradient of 0-0.2 MNaCl. The inset shows SDS-PAGE analysis of fractions 30 and 31, whichwere identified as PEB3 containing fractions by N-terminal sequencing ofSDS-PAGE bands.

FIG. 2. ESI-MS analysis of fractions from cation exchange chromatographypurification of PEB3. a) ESI-MS of the fraction 31 of FIG. 1, afterdialysis and the reconstructed molecular mass profile obtained from thisspectrum. b) The peaks at 25,454 and 28,376 Da were identified as PEB3and PEB4, respectively. Further analysis was required to identify thethird peak at 26,861 Da as glycosylated PEB3. c) ESI-MS of PEB3similarly purified from the pglB mutant, and the reconstructed massprofile, d) showing the lack of the glycosylated form of PEB3.

FIG. 3. MS/MS analysis of the PEB3 tryptic glycopeptide. a) Product ionspectrum of the doubly protonated glycopeptide ion at m/z 1057.9. Thefragment ions originating from the sequential loss of oligosaccharideresidues are indicated in the spectrum. The peptide sequence is shown inthe inset. b) Second generation product ion spectrum of the glycopeptidefragment ion at m/z 937.4. The glycopeptide was fragmented by front endcollision induced dissociation (orifice voltage=100 V) as it entered themass spectrometer. The observation of the b₃+228 fragment ion at m/z605.3 confirmed that the oligosaccharide is N-linked.

FIG. 4. 2D gels of the SBA affinity chromatography product. The proteinswere separated on 2D-PAGE in two pH ranges, a) pH 4-7 and b) pH 6-11,and then silver-stained. The identities of the spots, shown by their Cjnumbering, were determined by mass spectrometry of their trypticdigests. A full list of the identified proteins is given in Table I.

FIG. 5. Purification of glycopeptides from a pronase digest of the SBAaffinity chromatography product. a) Size exclusion chromatography onBioGel P4 200 mesh of the pronase digest. b) Re-fractionation of pooledmaterial from P4 on BioGel P2 fine grade. In both fractionations,glycoprotein-containing fractions were identified by MS and pooled asindicated by the bars. c) ESI-MS spectrum of fraction 10 from B above.The doubly protonated ion (MH₂ ²⁺) at m/z 770.5 corresponds to theheptasaccharide linked to Asn. A number of larger ions are also observedand are due to the addition of a second amino acid residue. The aminoacid compositions of the major glycan-containing ions are indicated onthe spectrum.

FIG. 6. Structure and ¹H spectra of the glycopeptide. a) Deducedstructure of the glycopeptide. b) Spectrum after final purification on aP2 column (D₂O, 35° C., 1 mM deuterated EDTA). The anomeric sugarresonances in the region 4.4 to 5.4 ppm are labeled.

FIG. 7. 1 D selective NMR experiments with the glycopeptide. a) 1DTOCSY(g1f2, 15 Hz, 144 ms), b) 1D TOCSY(e1f1, 25 Hz, 144 ms), c) 1DTOCSY(d1, 20 Hz, 144 ms), d) 1D TOCSY(b6, 50 Hz, 66 ms) where * is apeptide resonance, e) 1D TOCSY(b1c1, 40 Hz, 151 ms), f) 1D TOCSY(a1, 25Hz, 144 ms), g) 1D NOESY(g1f2, 15 Hz, 400 ms), h) 1D NOESY(f1, 10 Hz,400 ms), i) 1D NOESY(e1, 10 Hz, 400 ms), j) 1D NOESY(d1, 20 Hz, 400 ms),k) 1D NOESY(b1c1, 40 Hz, 400 ms), l) 1 D NOESY(a1, 40 Hz, 400 ms).

FIG. 8. The pgl locus and characterization of the pglB mutant. a) Geneschematic of the general protein glycosylation locus of C. jejuni NCTC11168. Genes which have homologues to genes in the pgl locus ofNeisseria spp. are shown by grey arrows. The mutation in pglB shownbelow the locus was constructed using pEAp26. b) to e) Two dimensionalgel analysis of C. jejuni wildtype and isogenic pglB mutant. b) and d)Colloidal coomassie stain of 2D gels before immunoblotting of wildtypeand mutant, respectively. c) and e) Immunodetection of proteins by HS:2serotyping sera of wildtype and mutant, respectively. The arrowsindicate the proteins which showed differences in either gel migrationand/or immunoreactivity which were excised for identification by massfingerprinting. The identities of the spots are shown by their Cjnumbering. The masses of the molecular weight protein markers in kDa areshown at the left.

FIG. 9. Detection of the N-linked glycan in the HR-MAS proton NMRspectra from various campylobacter strains. The structure of theN-linked glycan is shown above the spectra. a) Spectrum of the purifiedN-linked glycan in C. jejuni NCTC 11168 showing the anomeric resonanceslabeled a to g. HR-MAS NMR spectra using a 10 ms CPMG filter of wholecells of b) C. jejuni NCTC11168, c) C. jejuni NCTC11168 kpsM-, d) C.jejuni HS:19 serostrain, e) C. coli HS:30 serostrain and f) C. jejuniNCTC11168 pglB-. Common resonances in b) to e) compared to those in a)are indicated by vertical dotted lines. The HOD resonance at 4.8 ppm wassaturated and digitally filtered.

FIG. 10. Comparison of selective NMR experiments of C. jejuni HS:19cells and the purified N-linked glycan. HR-MAS spectra of C. jejuniHS:19 (a), c), e), g)) and NMR spectra of the purified N-linked glycan(b), d), f), h)). a) NOESY[a1, 90 Hz, 250 ms]. b) NOESY[a1, 40 Hz, 400ms]. c) NOESY[b1c1, 60 Hz, 250 ms]. d) NOESY[b1c1, 40 Hz, 400 ms]. e)NOESY[d1e1f1, 90 Hz, 250 ms]. f) Sum of NOESY[d1, 20 Hz, 400 ms] and ofNOESY[e1f1, 25 Hz, 400 ms]. g) TOCSY[g1f2, 72 Hz, 47 ms]. h) TOCSY[g1f2,15 Hz, 33 ms].

FIG. 11. The spectrum of the purified N-linked glycan in C. jejuniNCTC11168 showing the anomeric resonances labeled a to g with commonresonances indicated by vertical dotted lines is shown for comparison.

FIG. 12. High resolution magic angle spinning NMR with and withoutcobalt chloride to demonstrate surface exposure of the N-linked Pglglycan.

DETAILED DESCRIPTION OF THE INVENTION

The examples describe the isolation and the identification of the glycanmoiety according to the present invention.

General Experimental Procedures

Bacterial strains and plasmids: C. jejuni NCTC 11168 was routinely grownon Mueller Hinton agar under microaerophilic conditions (10% CO₂, 5% O₂,85% N₂) at 37° C. E. coli DH10B (Invitrogen) was used as the host strainfor cloning experiments and clones were grown on Luria S-gal agar(Sigma) or MH agar at 37° C. When appropriate, antibiotics were added tothe following final concentrations: kanamycin 30 μg/mL and ampicillin150 μg/mL. Plasmid pPCR-Script Amp (Stratagene) was used as the cloningvector.

Preparation of glycoprotein extracts: Cells from two plates of overnightgrowth were re-suspended in 10 mL Mueller Hinton broth and used toinoculate 1 litre of MH culture medium. Cultures were grown undermicroaerophilic conditions at 37° C. for 24 h with shaking at 150 rpm.Bacterial cells from 12 litres of culture media were harvested bycentrifugation at 10,000×g for 15 min and immediately frozen at −75° C.Frozen cell pellets were thawed on ice in 0.2 M glycine HCl buffer, pH2.2 (13), and extracted for 15 min with gentle stirring. Extracts wereclarified by centrifugation at 10,000×g for 15 min, dialyzed againstpure water (Milli-Q system, Millipore Corporation) and freeze-dried.

Purification and analysis of PEB3: The PEB3 protein was purified tohomogeneity by cation exchange chromatography of the glycine extract aspreviously described (7). A Pharmacia MonoS HR 5/5 column was used on anÅKTA Explorer LC system (Amersham Biosciences). The column eluate wasmonitored for UV absorbance at 280 nm and fractions were examined bySDS-PAGE analysis (21) in Mini Protean II slab gels (BioRadLaboratories). N-terminal sequencing of individual proteins wasperformed on a model 491 Procise protein sequencing system (AppliedBiosystems Inc.), following transfer from SDS gels to ProBlot™ PVDFmembrane (Applied Biosystems Inc.) as described by LeGendre et al. (22).

The protein molecular weight profiles of selected fractions weredetermined by electrospray ionization mass spectrometry using an AppliedBiosystems/Sciex Q-Star hybrid quadrupole time-of-flight massspectrometer. The fractions were first dialyzed extensively to removesalts, and adjusted to 30% methanol, 0.2% formic acid. The solution wasinfused at a flow rate of 1 μL/min and spectra were acquired over therange m/z 600>2000.

Analysis of tryptic peptides: Selected fractions were digested overnightat 37° C. with modified trypsin (Promega) in 50 mM ammonium bicarbonateand analyzed by capillary LC-tandem mass spectrometry using a capillaryHPLC system (CapLC, Waters) coupled with a Q-TOF2 hybrid quadrupoletime-of-flight mass spectrometer (Micromass). Approximately 250 ng ofeach digest was injected on a 0.3×150 mm PepMap C₁₈ capillary LC column(Dionex/LC-Packings) and resolved by gradient elution (5-90%acetonitrile, 0.2% formic acid in 45 minutes). The mass spectrometer wasset to operate in automatic MS/MS acquisition mode and spectra wereacquired on doubly, triply and quadruply charged ions.

Larger scale separation of the tryptic digest was carried out on a 4.6mm×250 mm Jupiter C₁₈ LC column (Phenomenex Inc.). The fractioncontaining the glycopeptide was then infused at a flow rate of 1 μL/mininto the microelectrospray interface of the Q-TOF2 mass spectrometer.Fragmentation of the glycopeptide prior to MS/MS analysis was achievedby front-end collision-induced dissociation (the orifice voltage wasincreased to 100 V from the normal 40 V). The MS/MS collision offset forthe singly charged fragment ions produced in this manner was 20-25 V(lab frame of reference). For β-elimination experiments by the method ofRademaker et al. (23), approximately half of the glycopeptide-containingfraction was evaporated to dryness and dissolved in 25% aqueous ammoniumhydroxide. The solution was left at room temperature overnight,evaporated to dryness for a second time and re-dissolved in water. Thesolution was then examined by infusion-MS as described above.

Purification and analysis of total glycoproteins: The glycoproteins fromthe glycine extracts were isolated by affinity chromatography on SBAlectin/agarose (Sigma-Aldrich Ltd.). The freeze-dried glycine extractwas redissolved in PBS (100 mM NaCl, 50 mM sodium phosphate pH 7.5) andpassed through an SBA/agarose column previously equilibrated in PBS. Thecolumn was washed with 10 column volumes of PBS and bound glycoproteinwas eluted with 0.1 M GalNAc in PBS. Glycoprotein-containing fractionswere pooled, dialyzed against Milli-Q water and freeze-dried.

The glycoproteins were separated by SDS-PAGE on 12.5% homogeneouspolyacrylamide gels (21). 2D-PAGE was performed using pre-cast IEFstrips containing immobilized linear pH gradients of either pH 3-10, pH4-7 (BioRad Laboratories) or pH 6-11 (Amersham Biosciences). Proteinswere solubilized in sample buffer according to the manufacturer'sinstructions and resolved by isoelectric focussing on the pre-cast IEFstrips followed by SDS-PAGE on homogenous 12.5% slab gels, 20×20 cm, forthe second dimension. Gels were stained with Bio-Safe colloidal G-250Coomassie blue stain (BioRad Laboratories) or silver stained (23). Forsubsequent lectin probing, the gels were electroblotted onto PVDFmembrane at 50 V for 1 h in 10 mM 3-(cyclohexylamino)-1-propanesulphonic acid buffer, pH 11, containing 10% methanol. The membrane waswashed in Milli-Q water and blocked in Tris-buffered saline (100 mMNaCl, 50 mM Tris pH 7.5) with 0.05% Tween 20 and 2% blocking buffer(Roche Molecular Biochemicals) for 2 h at room temperature. Followingblockage, blots were further incubated with SBA-alkaline phosphataseconjugate (EY Laboratories Inc.) at a concentration of 10 μg/mL in theabove blocking solution for 1 h at room temperature. Blots were washedthree times in Tris-saline with 0.05% Tween 20 and developed using nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate(BCIP) in 0.1 M NaCl, 0.1 M Tris pH 9.5 with 50 mM MgCl₂.

MS analyses of 2D-gel spots: The protein spots were excised anddestained with a 1:1 ratio of 30 mM potassium ferricyanide and 100 mMsodium thiosulfate (24). The gel spots were washed extensively withdeionized water, shrunk with acetonitrile, re-swollen with 50 mMammonium bicarbonate containing Promega modified trypsin (10 ng/μL) andsufficient 50 mM ammonium bicarbonate was added to cover the gel pieces(typically 30 μL). The tubes were sealed and incubated overnight at 37°C. The digest solutions were removed and the gel pieces were extractedwith 50 μL of 5% acetic acid and then with 50 μL of 5% acetic acid in50% aqueous methanol. The extracts were pooled with the digest solutionsand concentrated to approximately 10 μL.

The peptide extracts from the intense protein spots were analyzed byMALDI-TOFMS using a MALDI-LR mass spectrometer (Micromass).Approximately 0.5 μL of the MALDI matrix solution (10 mg/mLα-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile, 0.2% TFA) wasdeposited on the target plate and allowed to dry. The peptide extractswere desalted using C₁₈ ZipTips™ (Millipore) and were deposited directlyon the matrix spots. Acquisition of the MALDI-TOFMS spectra was carriedout automatically. The spectra were calibrated externally using peptidestandards and internally with trypsin autolysis peptides. Databasesearching was carried out in batch mode using Mascot Daemon™ (MatrixScience) and against the C. jejune NCTC11168 genome sequence database.

The extracts from the fainter protein spots were analyzed bynanoLC-MS/MS using the Q-TOF2 mass spectrometer. The entire samples wereinjected onto a 0.3×5 mm C₁₈ micro pre-column cartridge(Dionex/LC-Packings). The peptides were retained while the samplesolution was washed to waste. The trap was then brought online with a 75μm×150 mm C₁₈ Nano-Series column (Dionex/LC-Packings) and the peptideswere separated with a gradient supplied by the CapLC pump (15-75%acetonitrile, 0.2% formic acid in 30 minutes, approximately 300 nL/minflow rate). The mass spectrometer was set to acquire MS/MS spectra inautomated mode as described above. Database searching was carried out asdescribed for the MALDI-TOFMS analyses.

Glycopeptide preparation: Freeze-dried total glycoprotein (5 mg) wasdissolved in 250 μL of 100 mM Tris pH 8.0 containing 2 mM CaCl₂, anddigested with pronase as previously described (31). The digest wasmicrofuged at 10,000×g for 15 min and the supernatant was applied to acolumn (1×120 cm) of BioGel P4, 200 mesh (BioRad Laboratories). Thecolumn was run in water and the column eluate was monitored byrefractive index. Fractions were screened by ESIMS and precursor ionscanning mass spectrometry (precursors of the HexNAc oxonium ion at m/z204) on an API 3000 triple quadrupole mass spectrometer (AppliedBiosystems/Sciex). Fractions giving the HexNAc ion signature were pooledand freeze-dried. The glycopeptides were further purified on a 1×120 cmcolumn of BioGel P2 fine grade, the fractions being monitored andscreened as described above.

NMR spectroscopy: All spectra were acquired using a Varian Inova 600 MHzspectrometer using the standard Varian software. A gradient 4 mmindirect detection high-resolution magic angle spinning nano-NMR probe(Varian) with a broadband decoupling coil was used. The sample was spunat 3 KHz with dry nitrogen as the drive and bearing gas. The spin ratewas not computer controlled but remained constant to within 10 Hz of theset value. Samples in 40 μL D₂O solution were recorded at 25° C. and at35° C. to produce sharper peaks. The pH was unknown due to the smallvolume. Deuterated EDTA (CDN Isotopes Inc.) was added to chelate metalions and provide sharper peaks for bacillosamine and amino acids.Although the glycopeptide isolate from a P4 column contained some aminoacid and sugar impurities, spectra were of sufficient quality to allowcomplete resonance assignments of the glycopeptide in the presence of 15mM of deuterated EDTA. Much of the NMR structural work proceeded withthis sample because of the risk of loosing the bulk of the isolatedglycopeptide by further purification. The derived structure wasconfirmed by additional NMR experiments on glycopeptide that had beenpurified using a P2 column, lyophilized, and dissolved in 40 μL D₂O with1 mM deuterated EDTA. The experiments were performed with suppression ofthe HDO signal at 4.78 ppm (25° C.) and 4.67 ppm (35° C.). Acquisitionand processing of two-dimensional experiments (COSY, TOCSY, NOESY, HMQC,HMBC) were performed as described previously (32). The ¹H reference wasset by external acetone at 2.23 ppm. The ¹³C reference was set with themethyl resonance of external acetone at 31.07 ppm. The ¹H and ¹³Cchemical shifts in Table 1 were measured from the proton spectra andfrom C-H cross peaks in the HMQC and HMBC spectra. 1D TOCSY experimentswith various spin-lock times from 30-151 ms and 1 D NOESY with mixingtimes from 400-800 ms were performed as described previously (25,26).Selective experiments were described as 1D EXP(selected spins, selectiveexcitation bandwidth, mixing time) where EXP is TOCSY or NOESY.

The use of magic angle spinning (MAS) for liquid state samples in thepresence of both RF and magnetic-field homogeneities has been shown toinfluence significantly the performance of mixing sequences in TOCSYexperiments and can degrade performance (27,28). Using adiabatic (WURST)mixing sequences can eliminate such effects (27,29). The standard 2DTOCSY and 1D TOCSY sequences were modified so that the MLEV-17 orDIPSI-2 mixing sequence was replaced with the adiabatic WURST-2 pulses.The adiabatic (WURST-2) mixing had a single adiabatic inversion pulselength of T_(p)=1/MAS spin rate, a modulation depth of 8 and anadiabicity of 2. Typically, for the WURST-2 pulse, the sweep bandwidthwas 24 kHz, T_(p)=0.333 ms (at a MAS spin rate of 3000+/−10 Hz),B₁(max)=8.51 kHz, B₁ (RMS)=4.77 kHz.

GC-MS analysis: The enantiomeric configurations of the Glc and GalNAccomponents of the P2 product were assigned by characterization of thebut-2-yl glycosides in gas liquid chromatography-mass spectrometry (21).The derivatives were analyzed using a Hewlett-Packard chromatographequipped with a 30 m DB-17 capillary column (180° C. to 260° C. at 3.5°C./min), and spectra in the electron impact mode were obtained with aVarian Saturn II mass spectrometer.

Construction and characterization of pglB mutant: For construction ofthe pglB mutant, genes Cj1121c to Cj1126c were PCR amplified from C.jejuni NCTC 11168 using the primers: Cj1121cF(5′-ACTCACTATTGCCATTAAGATAAGC-3′) and Cj1126cR(5′-AAAACCCTTATTTAGTTTTGTTTGC-3′). The PCR product was polished with Pfupolymerase and then ligated into pPCR-Script Amp (Stratagene) accordingto the manufacturer's instructions. The ligation mixture waselectroporated into electrocompetent E. coli DH10B and selected for onLB S-gal agar (Sigma-Aldrich) with ampicillin. A blunt-ended kanamycinresistance cassette from pILL600 (37) was inserted into the filled-inXbal restriction site of pglB, generating pEAp26. The orientation of thecassette was determined by sequencing with the ckanB primer(5′-CCTGGGTTTCAAGCATTAG-3′). DNA was sequenced using terminatorchemistry and AmpliTaq cycle sequencing kits (Applied Biosystems) andanalysed on an Applied Biosystems 373 DNA sequencer. The mutated plasmidDNA was used for electroporation into C. jejuni NCTC 11168 (32) and thekanamycin-resistant transformants were characterized by PCR to confirmthat the incoming plasmid DNA had integrated by a double cross-overevent.

Proteins were extracted from C. jejuni whole cells using 0.2 M glycineat pH 2.2 (10) and dialysed against water. Samples were analyzed by2D-PAGE using 11 cm pH 3-10 ReadyStrip IPG strips (BioRad Laboratories)and pre-cast 12×8 cm 8-16% gradient Criterion slab gels (BioRadLaboratories). Gels were stained with colloidal coomassie blue,photographed, and then partially destained by washing in water. Proteinswere transferred for 1 h at 207 mA onto PVDF membranes using aTrans-Blot SD Semi-Dry Transfer Cell (BioRad). After blocking overnight,membranes were probed with a 1:500 dilution of HS:2 serotyping serumfollowed by a 1:5000 dilution of goat anti-rabbit antiserum(Sigma-Aldrich), and developed with NBT/BCIP (Roche MolecularBiochemicals).

Experimental Procedures for the Detection of Glycans from CampylobacterCells

Bacterial strains and growth conditions: Campylobacter jejuni NCTC11168(HS:2) was isolated from a case of human enteritis (46) and latersequenced by Parkhill et al. (6). C. jejuni serostrains: HS:1 (ATCC43429), HS:2 (ATCC 43430), HS:3 (ATCC 43431), HS:4 (ATCC 43432), HS:10(ATCC 43438), HS:19 (ATCC 43446), HS:36 (ATCC 43456) and HS:41 (ATCC43460) were obtained from ATCC; C. jejuni HS:23 was obtained from Dr.Peggy Godschalk, Erasmus University Medical Center, Rotterdam; C. jejuniOH4382 and OH4384 were obtained from Health Canada; and C. coli HS:30(NCTC 12532) was obtained from NCTC. All campylobacter strains wereroutinely grown on Mueller Hinton agar (Difco) under microaerophilicconditions at 37° C. C. jejuni NCTC11168 mutants were grown on MuellerHinton agar with 30 μg/mL kanamycin.

Construction and characterization of site-specific mutations:Construction of the C. jejuni NCTC11168 kpsM (12) and pglB (13) mutantshas already been described.

Preparation of cells for HR-MAS NMR: C. jejuni overnight growth from oneagar plate (˜10¹⁰ cells) was harvested and suspended in 1 mL of 10 mMpotassium buffered saline (pH 7) made in D₂O containing 10% sodium azide(w/v). The suspension was incubated for 1 h at room temperature to killthe bacteria. The cells were pelleted by centrifugation (7 500×g for 2min) and washed once with 10 mM potassium buffered saline in D₂O. Thepellet was resuspended by adding 20 μL of D₂O and then 40 μL of thesuspension was inserted into the rotor for analysis.

HR-MAS NMR spectroscopy: HR-MAS experiments were performed using aVarian Inova 600 MHz spectrometer equipped with a Varian nano-NMR probeas previously described (12,13). Spectra from 40 μL samples were spun at3 KHz and recorded at ambient temperature (21° C.). The experiments wereperformed with suppression of the HOD signal at 4.8 ppm. Proton spectraof bacterial cells were acquired with the Carr-Purcell-Meiboom-Gill(CPMG) pulse sequence [90-(τ-180-τ)_(n)-acquisition] (34) to removebroad lines arising from lipids and solid-like material. The totalduration of the CPMG pulse (n*2τ) was 10 ms with τ set to (1/MAS spinrate). One-dimensional selective TOCSY experiments with variousspin-lock times from 30-150 ms and selective NOESY with mixing timesfrom 100-400 ms were performed as described previously (25,35). For useunder MAS conditions, the TOCSY sequences were modified so that theDIPSI-2 mixing sequence was replaced with the adiabatic WURST-2 pulses(13). Selective experiments were described as EXP[selected spins,selective excitation bandwidth, mixing time] where EXP is TOCSY orNOESY. Typically, proton spectra of bacterial cells could be obtainedusing 256 to 1024 transients (15 min to 1 hour). For the selectiveexperiments on the N-linked glycan resonances present as a minorcomponent in the bacterial cells, the time for each TOCSY and NOESYvaried from 1 to 8 hours.

EXAMPLES Example 1 Purification and Characterization of PEB3

PEB3 protein (Cj0289c) was identified in 2D gels of a glycine extract bypeptide mass fingerprinting, as a component of a group of spotsfocussing within a range of pH 9-10 (results not shown). PEB3 waspurified from the extract by cation exchange chromatography, andre-fractionated on the same column, using a shallower NaCl gradient,resulted in the PEB3 appearing in three fractions (FIG. 1). SDS-PAGEanalysis showed two bands, whose N-terminal sequences were determinedfollowing their transfer to a PVDF membrane. Ten cycles of sequencingidentified the lower mass species as PEB3 while the higher mass, moreabundant component, was also PEB3 with a minor sequence corresponding toPEB4 (Cj0596).

The mass spectrum and the reconstructed molecular mass profile forfraction # 31 are presented in FIG. 2 a) and b). Three peaks wereobserved in the reconstructed mass profile. The peaks at 25,454 Da and28,376 Da correspond well with the expected molecular masses of PEB3(25,453 Da, Cj0289c) and PEB4 (28,377 Da, Cj0596) respectively, withoutsignal peptides. To identify the protein of mass 26,861 Da, CapLC-MS/MSanalysis was carried out on the tryptic digest of this fraction. All butone of the peptides identified could be assigned to PEB3 or PEB4, inaccord with the N-terminal sequence data. MS/MS analysis of theunidentified ion (FIG. 3 a) clearly identifies it as a glycopeptide. Afragmentation series composed of sequential losses of HexNAc (203 Da)and a single Hex (162 Da) can be observed in this spectrum. The trypticpeptide was identified as ⁶⁸DFNVSK⁷³ from PEB3. The residue mass of theoligosaccharide portion of this glycopeptide is 1406 Da, whichcorresponds well with the difference in the molecular weights of PEB3and the unknown protein peak observed in FIG. 2 b. Therefore, itappeared that approximately 50% of the PEB3 protein in this fraction wasmodified with a single oligosaccharide composed 5 HexNAcs, 1 Hex and anunusual sugar with a residue mass of 228 Da. Moreover, the MS/MSspectrum indicated that the oligosaccharide was linked to the peptidevia the 228 Da sugar moiety.

Example 2 Characterization of the Glycopeptide Linkage

The PEB3 tryptic peptide to which the oligosaccharide is attachedcontains sites for both N- and O-linkage, ie Asn and Ser. Therefore, itwas necessary to carry out further experiments to determine the natureof this linkage. Previously, we have used β-elimination to removeO-linked carbohydrates from the flagellin of C. jejuni 81-176 (5).However, this procedure failed to remove the oligosaccharide in thisinstance. This was our first indication that the oligosaccharide isN-linked. This was confirmed by MS/MS analysis of the singly protonatedfragment ion at m/z 937.0 produced by front-end collision-induceddissociation of the intact glycopeptide (FIG. 3 b). This ion is composedof the tryptic peptide plus the unusual 228 Da sugar only. An ion wasobserved at m/z 605.1 which could only be assigned to the b₃ fragmention plus the 228 Da sugar moiety. No fragment ions were observed tosuggest that the carbohydrate is linked to the serine residue. All ofthis evidence strongly suggests, that the oligosaccharide is linked tothe peptide at Asn70. Interestingly, this peptide contains theeukaryotic N-linkage consensus sequon, Asn-Xaa-Ser.

Example 3 Isolation and Identification of Glycoproteins

Putative glycoproteins were purified by SBA affinity chromatography fromthe glycine extracts of 40 g wet weight of cells. The yield of putativeglycoproteins was 5 mg as estimated by UV absorbance at 280 nm. TheGalNAc eluant was subjected to 1D- and 2D-PAGE (FIG. 4) and to ensurethat the proteins purified in this manner possessed lectin bindingproperties, rather than non-specific binding characteristics, westernblotting with an SBA/alkaline phosphatase conjugate was also carriedout. Approximately 13 protein species were visualized following 1DSDS-PAGE but this number increased substantially when the product wasanalyzed by 2D-PAGE. The proteins in individual bands from 1D SDS-PAGEand spots from 2D-PAGE were identified by mass fingerprinting anddatabase searching (Table I). Among the identified proteins are PEB3(Cj0829c) and CgpA (Cj1670c) previously identified by Linton et al. (8).The vertical pattern of spots with identical pls displayed by Cj1670c,and other proteins, likely indicates varying degrees of glycosylationsince examination of their predicted amino acid sequences, derived fromthe whole genome sequence of C. jejuni NCTC 11168 (6), revealed thepresence of multiple potential N-linked glycosylation sites containingthe sequon Asn-Xaa-Ser/Thr (Table I). In fact, MS/MS analysis of theCj1670c-containing in-gel digest extracts indicated that 3 of its 6N-linkage sites are occupied to varying extents (three Cj1670cglycopeptides were detected by capLC-MS/MS: ⁷TDQNITLVAPPEFQKEEVK²⁵,⁷⁷VLDVSVTIPEKNSSK⁹¹ and ⁹²QESNSTANVEIPLQVAK¹⁰⁸. A single glycopeptidewas also observed for Cj0114 (⁷¹LSQVEENNQNIENNFTSEIQK⁹¹) and for Cj0200c(¹DSLKLEGTIAQIYDNNK¹⁷). Furthermore, the mass and composition of theglycan component of all these glycopeptides appears to be identical tothat observed for PEB3.

However, certain proteins identified from the 2D-PAGE, notably Cj0147c,Cj0169, Cj0332c, Cj0334, Cj0638c, Cj1181c and Cj1534c do not contain anyof these specific sequons in their amino acid sequences. These proteinseither are non-covalently associated with SBA-binding proteins or bindnon-specifically to the column. This conclusion is supported by thefailure of these protein spots to react with the SBA/alkalinephosphatase conjugate following 2D PAGE and electroblotting (Table I).

Example 4 Preparation of Glycopeptides

The mixed glycoprotein sample was subjected to two rounds of pronasedigestion and the products were separated by gel filtration on BioGel P4(FIG. 5 a). The carbohydrate-containing fractions were located by massspectrometry and, after NMR studies (see below), the sample wasre-purified on BioGel P2 (FIG. 5 b). The final purified sample wascomposed mainly of the oligosaccharide linked to a single Asn (FIG. 5c). There was no evidence of any variation in the glycan component. Theyield of glycopeptides was estimated at around 200 μg.

Example 5 Preparation of Isolated Heptasaccharide

The heptasaccharide moiety identified in Example 4 (Formula I) may beobtained in isolation by known methods. For example, the amino acidmoiety (Asn) may be cleaved from the oligopeptide prepared in Example 4by enzymatic or chemical hydrolysis well known in the art. It may benoted that although the present inventors have not carried out the abovestep, it would be evident that such a step may be carried out to isolatethe heptasaccharide. The heptasaccharide can also be obtained directlyfrom the glycoprotein without going through any glycopeptide. Thiscleavage can also be performed by enzymatic or chemical hydrolysis. Forexample Patel et al. (41) teach the use of hydrazine for such cleavage.

Example 6 NMR Spectroscopy

With the use of selective methods, it was possible to work with theimpure P4 sample. The complete resonance assignment of the sugar moietywas done with this sample for fear of loss of glycopeptide upon furtherpurification. After a second purification using a P2 column, 25% of theglycopeptide was lost as judged by measurement of the S/N ratio for theP4 and P2 purified samples. An HMQC experiment was rerun on the P2purified sample to confirm the assignments obtained using the P4purified sample. From mass spectrometry results, the glycopeptide wascomposed of 5 HexNAc residues, a Hex residue, and an unknown sugar witha mass of 228 Da. The absolute configuration of the HexNAc and Hexresidues was determined to be D by chemical analysis. Analysis of the ¹Hand ¹³C NMR data indicated the presence of 8 anomeric protons labeled inalphabetical order (FIG. 6). A series of 1 D TOCSY for the anomericresonances was done for proton assignments (FIG. 7). Different mixingtimes were used to assign the spins within each residue. HMQC and HMBCwere then used to assign the ¹³C resonances. The NMR assignments aregiven in Table II. 1D NOESY experiments (FIG. 7) were used to obtain thesequence as shown (FIG. 6).

Residue g was assigned to β-D-Glcp. For the 1D TOCSY of g1 with a mixingtime of 144 ms (FIG. 7 a), all spins up to the H6 and H6′ resonanceswere detected, indicative of the large J_(H,H) couplings typical ofβ-glucopyranose. Resonance g1 also overlapped with f2, allowingdetection of the f1 to f4 resonances. Residue g was terminal due tosimilar ¹³C and ¹H chemical shifts for C2 to C6 with those ofβ-D-glucopyranose (36).

Five residues (a, c, d, e, and f) were identified as α-D-GalpNAc. Avalue of J_(1,2) of 3.6+/−0.2 Hz, the strong H1-H2NOE (FIG. 7), andJ_(H,H) coupling pattern which included a small coupling to H4 (FIG. 7)showed that these units had the α-D-galactopyranosyl configuration.Although, the e1 and f1 anomerics resonances could be selectivelyexcited with a narrow bandwidth of 10 Hz, the spectra for simultaneousexcitation of e1 and f1 are shown in FIG. 7 b. Resonances f2 to f4 werealso detected for the 1D TOCSY of g1f2 (FIG. 7 a). A chemical shift forC2 near 51 ppm was indicative of an acetamido group. The (C2, H4) HMBCcorrelations were used to assign the C2 resonances of the five GalNAcresidues. The 1D TOCSY-NOESY(H1, H4) was used to detect the NOE betweenH4 and H5 and thus assign the H5 resonances (25). The 1 D-TOCSYexperiments on the H5 resonances were then used to detect the H6sresonances (not shown). Integration of the P2 purified sample (FIG. 6 b)indicated 7 NAc groups, five of those corresponding to the five GalNAcresidues. Comparison of the ¹³C chemical shifts of the GalNAc units withthose of α-D-GalpNAc indicated that residues a, c, e, f were linked atO-4 due to downfield shifts for C4 (37). Residue f had a branch point atO-3 as established by a downfield shift for C3. Residue d was terminaldue to similar ¹³C chemical shifts for C2 to C6 with those ofα-D-GalpNAc (37).

Residue b was assigned to β-D-bacillosamine(2,4-diacetamido-2,4,6-trideoxy-β-D-glucopyranose). For residue b and c,the anomeric resonances partially overlapped. For the 1D TOCSY of the b1and c1 resonances with a mixing time of 144 ms (FIG. 7 e), broadresonances could be identified up to a CH₃ resonance (b6) at 1.14 ppm.For the 1D TOCSY for b6 at 1.14 ppm, with a mixing time of 66 ms (FIG. 7d), peaks up to b4 were observed. A series of 1D TOCSY with differentmixing time from 30 ms to 144 ms were done to assign the peaks. Forresidue b, the broad peaks do not provide the coupling constants.However, for the 1D TOCSY of b1 with a mixing time of 144 ms, resonancesup to H6 could be observed, similar to the 1D TOCSY for H1 of β-Glcp(residue g). Hence, such efficient transfer was indicative of largecoupling constants typical of a β-glucopyranosyl configuration. Theb1-b3 and b1-b5 NOEs observed in FIG. 7 k were also typical of the βanomeric configuration. Chemical shifts for C2 and C4 at 55 and 58 ppmwere indicative of acetamido groups, in accord with the presence 7 NAcgroups in the structure (FIG. 6 a). The chemical shift for C1 at 79 ppmand H1 at 5.1 ppm indicated an N-linked anomeric similar to that foundfor β-GlcNAc-Asn (20). Comparison of the chemical shifts of residue bwith those of 2,4-diamino-2,4,6-trideoxy-β-D-glucopyranose found inother structures indicated that Bac was linked at O-3, the only possibleglycosidation site (38-40).

The absolute configuration of residue b was obtained by NOEs aspreviously described for another structure containing bacillosamine(38). A strong NOE observed between the CH₃ resonance at 1.14 ppm (b6)and the NAc at 1.95 ppm was due to the close proximity of the CH₃ groupat C6 to the NAC-CH₃ group at C4. Since, the NAc resonance at 1.95 ppmwas isolated from the other NAC resonances it could be selectivelyexcited. A strong NOE was observed between this NAc resonance and theα-D-GalpNAc H1 resonance of residue a, due to the a(1-3)b linkage asshown below. This NOE can only occur if residue b has theD-configuration, where the NAc group at C4 of residue b is in closeproximity (3 Å) to the anomeric proton of residue a. This NOE is notpossible if residue b has the L-configuration, since the H1a/4b-NAcinterproton distances are greater than 5 Å.

The sequence was determined by 1D NOESY experiments (FIG. 7). The strongc1-f4 NOE and smaller c1-f6 and c1-f6′ NOEs established the c(1-4)flinkage. The strong d1-c4 NOE and a smaller d1-c6′ indicated the d(1-4)csequence. The e1-a4, f1-e4 and g1-f3 established the e(1-4)a, f(1-4)eand g(1-3)f linkages. The a1-b3 NOE (FIG. 7I), established the a-bsequence. The structure is shown in FIG. 6 a. The sequence is in accordwith the glycosidation shift observed for the linkage carbons (seeabove).

Example 7 Characterization of a pglB Mutant

A pglB mutant was constructed by cassette mutagenesis (FIG. 8). Glycineextracts of the mutant cells demonstrated dramatic changes in proteinimmunoreactivity by 2D-PAGE with western blotting with HS:2 serotypingsera (FIG. 8 b,e). Several of the proteins which showed a change in themobility and/or immunoreactivity on 2D gels were identified by massfingerprint analysis (Table I). The protein identifications are inagreement with those identified by SBA lectin affinity chromatography,providing further evidence that the proteins reactive with the GalNAclectin are glycosylated by the Pgl pathway. The full set of SBA-reactiveproteins was not observed in this experiment since it was performed onwhole glycine extracts and at different pH range, ie FIGS. 4 and 8 arenot directly comparable. In addition, PEB3 was purified from the pglBmutant by ion-exchange chromatography as described above, and analysisby mass spectrometry showed that the protein completely lacked theglycan (FIG. 2 c,d).

To show that the pglB mutation only affected the glycosylation of theglycoproteins, analyses were performed of the lipooligosaccharide andcapsular polysaccharide of the mutant. Deoxycholate-PAGE of proteinase Kdigests and mass spectrometry showed that the mass of the mutant LOScore was identical to that of the wildtype (results not shown). Inaddition, identical capsular repeats were visible in the extracts of thecells of the wildtype and the isogenic mutant on deoxycholate-PAGE. Tofurther demonstrate that the capsule was unaltered, we examined thepolysaccharide by HR-MAS NMR. The spectrum of the mutant was unchangedcompared to that of the wildtype (results not shown).

Example 8 Detection of N-linked Glycans in Whole Cells by HR-MAS NMR

In the HR-MAS NMR spectra of intact campylobacter cells (FIG. 9), a setof common ¹H resonances was detected. We previously determined thestructure of the N-linked glycan of C. jejuni NCTC 11168 using MS andnano-NMR techniques to be a heptasaccharide (13). As can be observed inthe HR-MAS spectra of C. jejuni NCTC11168, NCTC11168 kpsM-, C. jejuniHS:19 and C. coli HS:30, anomeric resonances which matched with those ofthe purified N-linked glycan were observed, suggesting that this glycanwas common to all (FIG. 9). In the spectra of NCTC11168 (FIG. 9 b),resonances corresponding to the N-linked glycan were less intense thanthe resonances from the CPS and some of the anomeric resonancesoverlapped. However, they could clearly be distinguished when thecapsular resonances were eliminated in the NCTC11168 kpsM mutant (FIG. 9c). The assignment of the common glycan resonances to the N-linkedglycan could be validated further by examining the spectra of theNCTC11168 pglB mutant in which protein glycosylation has been abolished(3, 13). As expected, the resonances of the N-linked glycan could not beobserved in the NMR spectrum of this mutant (FIG. 9 f).

The identity of the putative common glycan was further confirmed usingselective TOCSY and NOESY experiments (FIG. 10). Experiments werecarried out using C. jejuni HS:19 cells, since anomeric resonances ofthe N-linked glycan could be clearly observed. A standard selectiveNOESY experiment starting with selective irradiation of the a1 resonancein HS:19 established a correlation with a resonance at 4.21 ppm (FIG. 10a). This resonance has the same chemical shift as the a2 resonanceobserved in the NOESY spectra for the a1 resonance in the purifiedglycopeptide (FIG. 10 b). Comparison of the NOE patterns for otheranomeric resonances between HS:19 and the purified N-linked glycanclearly indicated a similar match between the two sets of experiments(FIG. 10 c-f). Since the NOE experiments reveal both intra- andinter-residue correlations, this agreement suggests that the sequence ofthe common glycan is identical to that of the purified N-linked glycanreported previously (13). A selective TOCSY experiment with irradiationof the g1 and f2 overlapping resonances at 4.5 ppm gave rise to crosspeaks corresponding to the g2 and f3 resonances (FIG. 10 g). Theirmultiplet shape and chemical shifts matched with those observed in thecorresponding experiment for the purified N-linked glycan (FIG. 10 h).Other resonances observed were due to partial excitation of the strongerCPS resonances. Hence, the selective excitation experiments performed onthe bacterial cells established the in situ identity of the commonN-linked glycan.

Detection of the N-linked glycan in the HR-MAS proton NMR spectra fromvarious Campylobacter species is shown in FIG. 11. The structure of theN-linked glycan is shown above the spectra. The spectrum of the purifiedN-linked glycan in C. jejuni NCTC11168 showing the anomeric resonanceslabeled a to g with common resonances indicated by vertical dotted linesis shown for comparison. HR-MAS NMR spectra using a 10 ms CPMG filter ofwhole cells of C. coli HS:30 (NCTC 12532, isolated from a pig inBrussels), C. coli 423 (isolated from a chicken in Alberta) and C. fetusssp. venerealis Biotype A (NCTC 10354 isolated from the vaginal mucus ofa cow) are shown. The HOD resonance at 4.8 ppm was saturated anddigitally filtered.

FIG. 12 shows high resolution magic angle spinning NMR with and withoutcobalt chloride to demonstrate surface exposure of the N-linked Pglglycan. The top spectrum in each is from samples in which CoCl₂ isadded. They have been processed identically. Note that the signal tonoise ratio (S/N) of the top spectrum is much less than that of thebottom spectrum in each. Since the spectra are acquired with effectivelya T2 filter, therefore lines that have been broadened by the CoCl₂ willappear weaker. Note that for the kspM mutant, peaks near 3.15 ppmexhibit very different relative intensities to the peak(s) near 3 ppm.The absolute S/N for the peaks near 3.15 ppm with/without CoCl₂ isapproximately the same while that for the peak that at 3 ppm is verydifferent. This indicates that the peaks at 3.15 ppm are most likelyinternal and not available to CoCl₂. Thus external residues aresignificantly reduced in intensity. In the case of HS:19, the sameobservation can be made for the 3.15 ppm versus 3 ppm peaks.Additionally, the aspartic acid peaks at ˜2.7 and ˜2.85 ppm (seeSzymanski et al, JBC, 2003) disappear in the present of CoCl₂ whichconfirms that residues exposed on the external surface or to the mediumare broadened. Since the N-linked Pgl glycan resonances (see Szymanskiet al, JBC, 2003 and FIG. 9) essentially disappear in the present ofCoCl₂, this indicates that they too are on the cell surface.

Preparation and Administration of Antibodies and Vaccines

Antibodies or antigen-binding fragments thereof that specifically bindto the glycan moiety according to the invention may be produced byconventional methods. For example, murine monoclonal antibodies may beraised against the glycan of Formula I (including fragments) optionallylinked to an oligopeptide or amino acid or immunogenic conjugate.Another strategy is to pan a pre-existing library of cloned genesderived from a camelid lymphocite and capable of expressing dAb antibodyfragments, to identify and isolate genes capable of expressing fragmentshaving immunogenic activity against the selected antigen. The gene(s)thus isolated may be are expressed in a bacteriophage library which hasbeen modified to contain this gene. Such an antigen may comprise theheptasaccharide described above or a fragment of such a heptasaccharide,optionally linked as described above to an amino acid, oligopeptide orother conjugate. This method is described in U.S. Pat. No. 5,759,808 toCasterman et al. A still further strategy is to incorporate the gene forexpressing the selected antibody or fragment into the genome of asuitable plant which may then serve as a livestock food source. Such aplant would then express the antibody or antibody fragment and whenconsumed by livestock would thus deliver a suitable dose of the antibodyor fragment to the animal.

It will thus be seen that a variety of methods exist to generateantibodies or fragments which specifically bind the glycans of thepresent invention. Such antibody fragments may be prepared or producedindependently of a complete antibody.

Antibodies may also be prepared by the traditional means of isolation ofsuch antibodies from serum of an animal which has been administeredsuitable doses of the antigen identified in the above examples. Forexample such methods are described in U.S. Pat. No. 5,759,808 toCasterman et al. This reference describes use of Camelid species toproduce a unique type of antibody that does not contain light chains.

Antibodies or fragments as described above may be administered with asuitable carrier to humans or livestock as form of passive immunization.When administered to livestock such as poultry the preferred approach isto provide the antibodies or fragments with feed or drinking water,whether by mixing in a concentrate or supplying a plant product which ismodified to express the antibody or antibody fragment. The antibodies orfragments when provided separately may be supplied with suitablecarriers as a concentrate for mixing with the feed or drink prior touse.

Such antibodies or fragments administered to livestock combat thepresence of campylobacter in the livestock, thus preventing thelivestock from spreading the pathogens to humans who eat or handle theanimal products, as well as protection of groundwater from contaminationby animal wastes as described above.

These antibodies and fragments may also be used to diagnose the presenceof campylobacter bacteria in humans or animals by conventional means. Aswell, they may also be used as a component of an assay to determine topresence of campylobacter in a sample such as water, soil or manure.

The heptasaccharide or fragments, optionally linked to an amino acid,oligopeptide or other suitable conjugate, may also be combined withsuitable adjuvants and immunostimulants for administration as a vaccine.For such a purpose, one or more suitable dosages are administered in aconventional manner.

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The abbreviations used herein are:    Bac, bacillosamine, 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose;    capLC-MS/MS, capillary liquid chromatography-tandem mass    spectrometry;    CE, capillary electrophoresis;    CPMG, Carr-Purcell-Meiboom-Gill;    CPS, capsular polysaccharide;    COSY, correlated spectroscopy;    DIPSI-2, decoupling in the presence of scalar interactions;    ESI-MS, electrospray ionization mass spectrometry;    GBS, Guillain-Barré Syndrome;    HMBC, heteronuclear multiple-bond correlation;    HMQC, heteronuclear multiple quantum correlation;    HR-MAS, high-resolution magic angle spinning;    LOS, lipooligosaccharides;    LPS, lipopolysaccharide;    MALDI-TOFMS, matrix assisted laser desorption time-of-flight mass    spectrometry;    MAS, magic angle spinning;    MLEV-17, Malcolm Levitt's decoupling cycle;    MS-mass spectrometry    MS/MS-tandem mass spectrometry    NOESY, nuclear Overhauser effect spectroscopy;    PVDF, polyvinylidene difluoride;    SBA, soybean agglutinin;    TOCSY, total correlation spectroscopy; and

WURST-2, wideband, uniform rate, and smooth truncation. TABLE IIdentification of proteins from 2-D gels Number of SBA pglB Cj GeneAnnotation sequons^(a) staining^(b) mutant^(c) Cj0114^(d) probableperiplasmic 2S, 3T + + protein Cj0143c periplasmic solute binding 1S,1T + protein for ABC transport system Cj0147c thioredoxin (TrxA) −Cj0169 superoxide dismutase (Fe; − SodB) Cj0175c^(e) putative ironuptake ABC 1S, 2T + + transport system periplas- mic iron bindingprotein Cj0200c^(d) probable periplasmic 1T + + protein Cj0238 probableintegral membrane 5S, 1T − protein Cj0289c^(d) major antigenic peptide2S + + (PEB3) Cj0332c nucleoside diphosphate − kinase (Ndk) Cj0334 alkylhydroperoxide − reductase (AhpC) Cj0376 probable periplasmic 1S, 1T +protein Cj0415^(e) putative oxidoreductase 3T + sub-unit Cj0420 probableperiplasmic 1T ± + protein Cj0493 translation elongation factor 1T ±EF-G (FusA) Cj0511 probable secreted 2S, 2T + + proteinase Cj0638cinorganic pyrophosphatase − (Ppa) Cj0694 probable periplasmic 4S, 2T +protein Cj0715 transthyretin-like peri- − + plasmic protein Cj0779thioredoxin peroxidase 1S + (Tpx) Cj0835c aconitate hydratase (AcnB) 1S,2T + Cj0843c^(e) putative secreted trans- 5S, 3T + glycosylase Cj0906cprobable periplasmic 2S, 2T + protein Cj0944c probable periplasmic 1S,1T + protein Cj0998c probable periplasmic 1S, 1T + + protein Cj1018cbranched-chain amino-acid 1S, 2T + ABC transport system peri- plasmicbinding protein Cj1032 probable membrane fusion 2T + component of effluxsystem Cj1181c translation elongation factor − EF-Ts (Tsf) Cj1214chypothetical protein 1S ± Cj1221 60 kD chaperonin (Cpn60; 1S, 2T +GroEL) Cj1345c probable periplasmic 5S, 2T + protein Cj1380 probableperiplasmic 2T − protein Cj1444c^(e) putative capsule poly- 3S, 2T +saccharide export system periplasmic protein (KpsD) Cj1496c probableperiplasmic 1S, 1T + + protein Cj1534c probable bacterioferritin −Cj1565c paralysed flagellum protein 5S, 2T + (PflA) Cj1643 putativeperiplasmic 3S, 1T + protein Cj1659 periplasmic protein (P19) −Cj1670c^(d) probable periplasmic 4S, 2T + + protein (CpgA)^(a)S, Asn-Xaa-Ser sequons; T, Asn-Xaa-Thr sequons.^(b)Reactivity with SBA in Western blots of 2D gels.^(c)Proteins with changed spot position and/or immunoreactivity in 2Dgels, in the pglB mutant. Cj1018c & Cj1643 were not isolated by SBAchromatography.^(d)Glycopeptides observed by CapLC-MS/MS.^(e)Identified from 1-D gel.

TABLE II Chemical shifts (ppm) of the C. jejuni Asn-linked glycopeptide.Measured at 600 MHz (¹H) in D₂O, 35° C. with HOD at 4.67 ppm. Externalacetone methyl resonance at d_(H) 2.23 ppm and d_(c) 31.07 pppm. Erroron d_(c) is ±0.2 ppm and ±0.02 ppm for d_(H). Seven NAc resonances atd_(H) 2.07, 2.05, 2.04, 2.03, 2.02, 2.02 and 1.95 ppm. NAc-CH₃ d_(c) at23.1-23.4 ppm. NAc-CO d_(c) at 175-176 ppm. C1 C2 C3 C4 C5 C6 Residue H1H2 H3 H4 H5 H6 H6′ (a) a-GalNAc 98.0 50.7 68.0 77.6 72.7 62.2 5.21 4.213.83 4.02 3.83 3.93 3.90 (b) β-Bac 79.0 54.5 76.3 58.1 75.0 17.6 5.113.91 3.91 3.80 3.59 1.14 (c) a-GalNAc 98.3 51.6 67.9 77.5 71.8 60.9 5.104.26 4.14 4.10 4.46 3.71 3.62 (d) a-GalNAc 99.7 51.4 68.4 69.6 72.3 60.75.04 4.20 4.01 4.03 4.36 3.67 3.61 (e) a-GalNAc 99.6 51.6 67.9 77.5 72.460.7 5.00 4.25 4.12 4.09 4.35 3.67 3.61 (f) a-GalNAc 99.9 50.7 77.5 75.872.5 60.5 4.98 4.50 4.14 4.32 4.42 3.61 3.53 (g) β-Glc 106.1 74.1 76.971.1 77.2 62.1 4.51 3.28 3.46 3.35 3.39 3.89 3.68

1. A compound comprising a heptasaccharide of formulaGalNAc-a1,4-GalNAc-a1,4-[Glc-β1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-Bac,wherein Bac is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose or animmunologically active fragment thereof.
 2. The compound as defined inclaim 1 linked to an amino acid or oligopeptide.
 3. The compound asdefined in claim 2 wherein said amino acid is asparagine.
 4. Thecompound as defined in claim 1 derived from a glycoprotein isolated andpurified from a campylobacter bacterium.
 5. The compound as defined inclaim 4, wherein said bacterium is selected from C. jejuni, C. coli andC. fetus ssp. venerealis.
 6. A method of detecting a glycan moiety of abacterial glycoprotein, the method comprising subjecting said sample tohigh resolution magic angle spinning nuclear magnetic resonance (HR-MASNMR) spectroscopy.
 7. A pharmaceutical composition comprising a compoundas defined in claim 1 and a physiologically acceptable carrier.
 8. Thepharmaceutical composition as defined in claim 7, further comprising animmunogenic conjugate.
 9. The pharmaceutical composition of claim 7 or8, further comprising an immunostimulant.
 10. Use of the pharmaceuticalcomposition as defined in any one of claims 7 through 9 as a vaccine inan animal or a human.
 11. An antibody or an antigen-binding fragment ofan antibody that specifically binds with a compound comprising a glycanof the formulaGalNAc-a1,4-GalNAc-a1,4-[Glc-β1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-Bac,wherein Bac is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose or animmunologically active fragment thereof.
 12. An antibody or anantigen-binding fragment of an antibody that specifically binds with acompound as defined in claim
 1. 13. An antibody or an antigen-bindingfragment of an antibody which specifically binds with a compound asdefined in claim 1 derived from a gene isolated from a camelid.
 14. Theantibody or antigen-binding fragment thereof as defined in claim 13,wherein said CAMELID is selected from Camelus bactriamus, Camelusdromaderius, Lama pPaccos, Lama ggGlama and Lama vVicugna.
 15. Apharmaceutical composition comprising the antibody or antigen-bindingfragment as defined in claim 11, and a physiologically acceptablecarrier.
 16. Use of the pharmaceutical composition as defined in claim15 as a therapeutic agent in a human or an animal.
 17. A method ofreducing the presence of campylobacter pathogens from livestock, themethod comprising administering to the livestock the antibody orantigen-binding fragment as defined in claim
 11. 18. A method as definedin claim 17, wherein said administration consists of feeding thelivestock with feed mixed with said antibody or antigen-bindingfragment.
 19. The method as defined in claim 17, wherein said livestockis poultry.
 20. A method of preventing campylobacter infections causedby campylobacter pathogens in a human, the method comprising removingsaid pathogens from livestock by the method as defined in claim
 17. 21.A method of treating a disease caused by campylobacter pathogens in ahuman or an animal, the method comprising administering the antibody orantigen-binding fragment as defined in claim
 11. 22. A method ofpreventing ground water contamination by campylobacter pathogens, themethod comprising reducing the presence of said pathogens from livestockby the method as defined in claim
 17. 23. An animal feed or drink forlivestock comprising the antibody or antigen-binding fragment as definedin claim
 11. 24. A feed or drink as defined in claim 23 comprising anadditive comprising said antibody or fragment.
 25. A feed as defined inclaim 23 comprising a plant containing a genome modified to express saidantibody or fragment.
 26. A plant genome containing a gene capable ofexpressing the antibody or antibody fragment as defined in claim
 11. 27.A plant cell containing the genome as defined in claim
 26. 28. A plantcontaining the cell as defined in claim
 27. 29. The plant genome asdefined in claim 26, wherein said gene is obtained by panning a libraryof camelid genes with a probe comprising the compound as defined inclaim 1, for camelid genes capable of expressing said antibody fragmentand incorporating said gene in a plant genome.
 30. A diagnostic kit fordetecting the presence of campylobacter in animals or humans, said kitcomprising the antibody or antibody fragment as defined in claim
 11. 31.A diagnostic kit for detecting the presence of campylobacter in asample, said kit comprising the antibody or antibody fragment as definedin claim
 11. 32. The kit as defined in claim 31 wherein said sample isselected from water, soil and manure.